circEXOC5 promotes acute lung injury through the PTBP1/Skp2/Runx2 axis to activate autophagy

circEXOC5 activates autophagy and promotes acute lung injury via stimulating the Skp2 decay to stabilize Runx2 by interacting with PTBP1.

In the manuscript by Gao et al., the authors examine the role of circRNA in promoting lung injury via the autophagy pathway. The authors find that when ALI is induced in their mouse model that levels of circEXOC5 increase, which correlates with increased pathology and markers of the autophagic pathway. shRNA depletion of circEXOC5 rescues this phenotype. Mechanistically, the authors implicate the protein PTBP1 and a potential involvement of Skp2/Runx2. Overall, the data are clear and the manuscript is well written. I do have some points below that should help to make the authors conclusions more robust. I also caveat my review with the fact that I am not familiar with circRNAs or ALI models.
Main points: 1) The authors only use 1 shRNA sequence targeting circEXOC5. Is there potential for off target effects and should this be controlled for?
2) Additional higher magnification TEM images are needed in Fig. 3B and 8D. Cells look very vacuolated, which appear empty and not autophagosome-like (double/multiple limiting membranes filled with cytosolic content). Are the authors sure that they are quantifying the correct organelles here?
3) Lung data is consistent with increased autophagy, but it could also equally mean that autophagy is inhibited -given it is a dynamic process and markers such as LC3 and p62 can go up or down if the rate of autophagosome formation relative to the rate of degradation changes. Hence it is essential to measure autophagy flux (normally by -/+ a lysosomal inhibitor). While this would be challenging to do in vivo, the authors could carry this out in MPVECs with their LPS treatments -/+ Bafilomycin A1 (not chloroquine as this also activates the CASM pathway). These assays are a standard in the autophagy field and would greatly help with the robustness of the authors' conclusions. 4) On page 14, and in relation to Fig. 4, I think the authors are a little misleading with their modulators of autophagy. While 3-MA will inhibit autophagy, it is not a specific autophagy inhibitor. It targets PI3Kinases, which affect a multitude of cellular process. Likewise, rapamycin inhibits mTORC1, which is again involved in many autophagy-independent processes. The authors should alter their text to reflect this by saying that their results are consistent with autophagy, but other process cannot be ruled out with these compounds given the multiple roles of their targets. Fig.6, what is the level of overexpression of PTBP1 in these experiments? The levels relative to empty vector alone treatments would be very helpful here to determine if they are close to endogenous or vastly higher.

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6) The data in Fig. 7 is intriguing but it is not clear what relevance Skp2/Runx2 has to ALI. Perhaps I am missing some relevant background information, as the authors do not show directly that Skp2/Runx2 levels influence the ALI phenotype, or indeed autophagy flux? Is it possible that Skp2/Runx2 could be mediating other functions of circEXOC5, which are not relevant to ALI? If so, perhaps these data could go as supplemental as potential candidates to check in future work (and change the title)? 7) On P17, first paragraph in relation to Fig. 7C, the authors state that simultaneous silencing of both circEXOC5 and PTBP1 fails to upregulate Skp2 -but the figure shows silencing of circEXOC5 and overexpression of PTBP1.Please clarify.
Minor points: 8) Expand abbreviations in first instance. 9) Molecular weight markers are needed on blots.
Reviewer #2 (Comments to the Authors (Required)): The authors explore the role of a circulating RNA (CircEXOC5) in the preventing the ALI development; they describe that autophagy is a well-known mechanism involved in ALI progress and demonstrate that circEXOC5 is a regulator of autophagy. The authors' purpose of the study is clear. The aim is presented clearly in the manuscript and the objectives of the study are well defined. The methods used to obtain the results are right to the purpose searched. The major asset of this manuscript is that it presents new data that can be used for new therapeutic approximations. These findings may have implications for the current research.
In my point of view, the manuscript is valuable for the scientific community and the current research in ALI, but I have some comments and concerns that I feel need to be addressed to make the manuscript suitable for its publication.
Introduction: •I miss a proper explanation of the circEXOC5 action mechanism and what is known in the literature. Please, use a paragraph to explain the circEXOC5/PTBP1/Skp2/Runx2 signaling cascade. •As afterward, autophagy is one of the main studied pathways, authors should mention in the introduction how autophagy is affected during ALI.
•Authors mention that circEXOC5 may have a biological significance in ALI treatment, however, I disagree with this affirmation. CircEXOC5 silencing was administered long before triggering ALI, so, it will be preventing ALI development/progress, but the authors cannot confirm a therapeutic effect of it, as it was not used as a treatment after the disease was triggered. This needs to be "corrected" throughout all the manuscript and discussed in the results section. Do the authors have data regarding a therapeutic (treatment after the disease/syndrome is established) effect of CircEXOC5 or downstream pathway effectors? Methods: •Which parental strain of mice did the authors use? C57BL/6J or C57BL/6N? I feel it is important to mention it because both strains have different inflammatory responses under the acute stimuli.
•Why did the authors decide to use male mice? Are the data reproducible in female mice? Please, include this information in your discussion as a limitation if you only used one sex, because data are not confirmed for both sexes. •In vivo, circEXOC5 was administered before triggering •Please, mention the abbreviations at least once before using them (OE=overexpression, no? but it is not mentioned in the methods the first time that it is used; the same applies for Lv, MA...)

Results:
•Authors used the term normal in figure 1 for animals without damage, are they the control for the sham (CLP surgery) and vehicle mice (for the Lv injection)? •Did the authors observe any other systemic effect due to the administration of circEXOC5? CLP-sepsis/ALI model is producing a systemic inflammation and other organs get affected due to it. Did the authors measure any circulation/systemic inflammatory marker such as IL6/IL1b in plasma? If yes, are they also mitigated by the knockdown of circEXOC5? If not, can you provide these data? •Did the authors observe any other organ affectation due to the circEXOC5 knocking down? Any effects on the liver or kidney? •Fig 2-C-D show isolated cells from BAF; however, these cell populations probably were quite different if isolated from an injured or non-injured animal; and they will have different responses and production of pro-inflammatory cytokines. However, the images look like the seeded cells were mainly macrophages (due to the shape). Can you indicate which cell population from BAF was used? Were they characterized? If not, how can you be sure that you are not comparing the response of a 40%macrophages: 60% neutrophil with a cell population of 90% macrophages? •Images 5F are not clear enough to confirm if the expression is intracellular/cytoplasm/membrane. Can you please provide higher magnification pictures with less brightness that allows us to confirm your statements? •Can you please explain/discuss for which mechanism LC3 is so strongly induced in figure 6B after shcircEXOC5 and OE-PTBP1? •In figure 7 looks like they are shcircEXOC5 and OE-PTBP1, however in the text authors mention that after silencing both failed to upregulate skp2 (which makes more sense). Please correct the disagreement between the text and the figure. Response: Thanks very much for your suggestion. We provide the summary blurb as follows: 'CircEXOC5 activates autophagy and promotes acute lung injury via stimulating the Skp2 decay to stabilize Runx2 by interacting with PTBP1.'.
--By submitting a revision, you attest that you are aware of our payment policies Reviewer #1 (Comments to the Authors (Required)): In the manuscript by Gao et al., the authors examine the role of circRNA in promoting lung injury via the autophagy pathway. The authors find that when ALI is induced in their mouse model that levels of circEXOC5 increase, which correlates with increased pathology and markers of the autophagic pathway. shRNA depletion of circEXOC5 rescues this phenotype. Mechanistically, the authors implicate the protein PTBP1 and a potential involvement of Skp2/Runx2. Overall, the data are clear and the manuscript is well written. I do have some points below that should help to make the authors conclusions more robust. I also caveat my review with the fact that I am not familiar with circRNAs or ALI models.
Main points: 1) The authors only use 1 shRNA sequence targeting circEXOC5. Is there potential for off target effects and should this be controlled for?
Response: Thanks very much for your suggestion. We originally designed two distinct shcircEXOC5 sequences (shcircEXOC5#1 and shcircEXOC5#2) and performed most experiments using both sequences. The data from both shcircEXOC5 sequences were nearly identical and thus we only presented those from shcircEXOC5#1 in the previous draft. In the revision, we added the data from shcircEXOC5#2 into Fig. 1 3) Lung data is consistent with increased autophagy, but it could also equally mean that autophagy is inhibited -given it is a dynamic process and markers such as LC3 and p62 can go up or down if the rate of autophagosome formation relative to the rate of degradation changes. Hence it is essential to measure autophagy flux (normally by -/+ a lysosomal inhibitor). While this would be challenging to do in vivo, the authors could carry this out in MPVECs with their LPS treatments -/+ Bafilomycin A1 (not chloroquine as this also activates the CASM pathway). These assays are a standard in the autophagy field and would greatly help with the robustness of the authors' conclusions.
Response: We sincerely appreciate the Reviewer's insightful suggestion. Following Likewise, rapamycin inhibits mTORC1, which is again involved in many autophagy-independent processes. The authors should alter their text to reflect this by saying that their results are consistent with autophagy, but other process cannot be ruled out with these compounds given the multiple roles of their targets. Reviewer #2 (Comments to the Authors (Required)): The authors explore the role of a circulating RNA (CircEXOC5) in the preventing the ALI development; they describe that autophagy is a well-known mechanism involved in ALI progress and demonstrate that circEXOC5 is a regulator of autophagy.
The authors' purpose of the study is clear. The aim is presented clearly in the manuscript and the objectives of the study are well defined. The methods used to obtain the results are right to the purpose searched. The major asset of this manuscript is that it presents new data that can be used for new therapeutic approximations.
These findings may have implications for the current research.
In my point of view, the manuscript is valuable for the scientific community and the current research in ALI, but I have some comments and concerns that I feel need to be addressed to make the manuscript suitable for its publication. •As afterward, autophagy is one of the main studied pathways, authors should mention in the introduction how autophagy is affected during ALI.
Response: Thanks very much for your suggestion. We added background information on autophagy and ALI in Paragraph 1 of the Introduction section.
•Authors mention that circEXOC5 may have a biological significance in ALI treatment, however, I disagree with this affirmation. CircEXOC5 silencing was administered long before triggering ALI, so, it will be preventing ALI development/progress, but the authors cannot confirm a therapeutic effect of it, as it was not used as a treatment after the disease was triggered. This needs to be "corrected" throughout all the manuscript and discussed in the results section. Response: Thanks very much for your suggestion. We only used male mice.
Following the Reviewer's comment, we have included using male mice only as a limitation for this study in the text (Discussion, the second to the last paragraph).
•In vivo, circEXOC5 was administered before triggering Response: Thanks very much for your suggestion. We have revised the manuscript at multiple places to suggest targeting circEXOC5 presented a preventive, instead of therapeutic effect in ALI. Please see the revised manuscript.
•Please, mention the abbreviations at least once before using them (OE=overexpression, no? but it is not mentioned in the methods the first time that it is used; the same applies for Lv, MA...) Response: Thanks very much for your suggestion. We have double checked all abbreviations and made corrections as needed. Please see the revised manuscript. Results: •Authors used the term normal in figure 1 for animals without damage, are they the control for the sham (CLP surgery) and vehicle mice (for the Lv injection)?
Response: Thanks very much for your suggestion. We have revised normal to sham in •Did the authors observe any other organ affectation due to the circEXOC5 knocking down? Any effects on the liver or kidney?
Response: We thank the Reviewer for this important comment. Unfortunately, we focused on lung injury in this study and did not examine the impacts of knocking down circEXOC5 on other organs. We have discussed this point as a potential caveat for this study (Discussion, the second to the last paragraph).
• Fig 2-C-D show isolated cells from BAF; however, these cell populations probably were quite different if isolated from an injured or non-injured animal; and they will have different responses and production of pro-inflammatory cytokines. However, the images look like the seeded cells were mainly macrophages (due to the shape). Can you indicate which cell population from BAF was used? Were they characterized? If not, how can you be sure that you are not comparing the response of a 40%macrophages: 60% neutrophil with a cell population of 90% macrophages?
Response: Thanks very much for your suggestion. We did isolate macrophages from BALF, which we provided more details in the Methods and Results section. Please see the revised manuscript.
•Images 5F are not clear enough to confirm if the expression is intracellular/cytoplasm/membrane. Can you please provide higher magnification pictures with less brightness that allows us to confirm your statements?
Response: Thanks very much for your suggestion. Following the Reviewer's suggestion, we have replaced the original Fig. 5F with images of higher magnification and improved clarity.
•Can you please explain/discuss for which mechanism LC3 is so strongly induced in figure 6B after shcircEXOC5 and OE-PTBP1? Thank you for submitting your revised manuscript entitled "CircEXOC5 promotes acute lung injury through the PTBP1/Skp2/Runx2 axis to activate autophagy". We would be happy to publish your paper in Life Science Alliance pending final revisions necessary to meet our formatting guidelines.
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